Method and Pharmaceutical Composition for Cartilage Regeneration

This application claims priority to U.S. Provisional Application No. 63/575,293, filed on Apr. 5, 2024, the content of which is hereby incorporated by reference in its entirety.

The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Apr. 2, 2025, is named “FEH0007US-Sequence Listing.xml” and is 8,128 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

The invention concerns a new method and pharmaceutical composition for cartilage regeneration.

Cartilage is a tissue composed of extracellular matrix and cartilage cells as chondrocyte, which has neither no blood vessels nor nerve. Therefore, it is difficult to heal the cartilage tissue when it is damaged. Further, chondrocytes are surrounded by hard extracellular matrix, and thus it is difficult to regenerate cartilage once damaged or degenerated.

The interactions of varieties of growth factors, cytokines, and signaling molecules have been reported to regulate the chondrogenic differentiation of mesenchymal stromal cells [1]. Transforming growth factor-beta (TGF-β) superfamily members is crucial for the in vitro chondrogenic commitment of mesenchymal stromal cells. However, the upregulation of TGF-β contributes to cell degeneration and inflammation that had been reported to interfere with chondrogenesis and has a risk of inducing fibrotic side effects [2-4]. On the other hand, accumulative findings from analysis of the expression of Wnt signaling molecules and in vivo and in vitro functional experiments suggest that inhibition of Wnt signaling is a therapeutic target for OA [5, 6]. However, the WNT inhibitors have been reported in the early clinical stages as an increase in bone remodeling with off-target side effects. It may imply that aberrant Wnt signaling would cause or be closely associated with skeletal disorders such as dwarfism, deformity of skeletons, osteoporosis, and high bone-mass syndrome [7].

Accordingly, it is desirable to develop a state-of-the-art molecule as a new therapeutic target that regulates chondrogenesis and cartilage repair in treating osteoarthritic articular cartilages.

Accordingly, the present invention provides a new approach to enhance the cartilage regeneration by using a protein or peptide, and thus which can be developed as a new drug for treating cartilage damages.

In one aspect, the present invention provides a method for cartilage regeneration in a subject, which comprises administering said subject a therapeutically effective amount of a composition comprising a CXCR4 agonist, wherein the agonist has a CXCR4 agonistic activity that induces chondrogenesis.

In one embodiment of the invention, the agonist is CXCL14 protein, or a variant, variable domain, functional derivative, or fragment thereof, which has the same function of CXCL14, a genetically modified recombinant protein or chemically modified protein thereof, or a gene construct for expressions of CXCL14.

In another embodiment of the invention, the agonist is CXCL12 protein, or a variant, variable domain, functional derivative, or fragment thereof, which has the same function of CXCL12, a genetically modified recombinant protein or chemically modified protein thereof, or a gene construct for expressions of CXCL12.

In one further aspect, the present invention provides a method for treating a cartilage defect disease in a subject, comprising administering said subject a therapeutically effective amount of a composition comprising a CXCR4 agonist, wherein the agonist has a CXCR4 agonistic activity that induces chondrogenesis.

In one embodiment of the invention, the cartilage defect disease is degenerative osteoarthritis.

In a yet aspect, the present invention provides a method for treating a cartilage defect disease in a subject, comprising administering said subject with a cell preparation obtained by culturing cells or stem cells treated with a CXCR4 agonist, wherein the agonist has a CXCR4 agonistic activity that induces chondrogenesis.

In a further aspect, the present invention provides a method for treating a cartilage defect disease in a subject, comprising administering said subject with a cell medium treated with a CXCR4 agonist, wherein the agonist has a CXCR4 agonistic activity that induce chondrogenesis.

In the present invention, the cell medium contains the secretome derived from the cells treated with a CXCR4 agonist.

In one yet aspect, the invention provides a method for treating a cartilage defect disease in a subject, comprising administering said subject with a cell preparation containing cells treated with extracellular vesicles (EVs) released from the cells treated with a CXCR4 agonist, wherein the agonist has a CXCR4 agonistic activity.

In one embodiment of the invention, the cells are stem cells, including induced pluripotent stem cells.

In one example of the invention, the stem cells are mesenchymal stem cells (MSCs), including but not limited to Wharton's jelly-derived MSCs (WJ-MSCs), infrapatellar fat pad-derived MSCs (IPFP-MSCs), subcutaneous adipose tissue-derived MSCs (SC-MSCs), amniotic fluid-derived MSCs (AF-MSCs), bone marrow-derived MSCs (BM-MSCs), umbilical cord-derived MSCs (UC-MSCs).

In the invention, the method for treating a cartilage defect disease in a subject, comprises a combination of two or more of the above-mentioned methods.

In one further yet aspect, the present invention provides a method for detecting a cartilage defect disease, wherein the gene expression signatures of CXCL14, CXCL12 or CXCR4 is used as a biomarker for treating a cartilage defect disease.

In one example of the invention, the CXCL14 protein (CXCL14-P) has the amino acid sequence of ITTKSVSRYRGQEH (SEQ ID NO:1).

In one example of the invention, the CXCL12 protein (CXCL12-P) has the amino acid sequence of RANVKHLKILN (SEQ ID NO:2).

In one example of the invention, the CXCR4 protein (CXCR4-P) has the amino acid sequence of MGYQKKLRSMTDKYRL (SEQ ID NO:3).

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

As used herein, the singular forms “a”, “an” and “the” include plural references unless explicitly indicated otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and their equivalents known to those skilled in the art.

As used herein, the term “cartilage” includes hyaline cartilage, fibrocartilage, or elastic cartilage, and is not particularly limited. One particular example in the present invention, is knee cartilage.

As used herein, the term “cartilage damage” refers to a disease caused by cartilage defects, injuries, damages, or damages caused by cartilage, cartilage tissue and/or joint tissues (synovial membrane, articular capsule, cartilaginous bone, etc.) injured by mechanical stimulation or inflammatory reaction. Such cartilage defect diseases include, but are not limited to, degenerative arthritis, rheumatoid arthritis, fractures, muscle tissue damage, plantar fasciitis, humerus ulcer, calcified myositis, or joint damage caused by fracture nonunion or trauma. In the invention, one particular example is osteoarthritis (OA).

As used herein, the phrase “therapeutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level may be determined by the species and severity, age, sex, type of disease, duration of treatment, factors including co-administered drugs, and factors well known in other medical disciplines.

As used herein, the term “subject” refers to a human or any animal, including a human or an animal particularly pet animals such as dogs, cats, horses and etc., with a cartilage damage, and indicates a group in need of a treatment for improving cartilage regeneration or a treatment of a cartilage damage.

As used herein, the term “Chemokine (C—X—C motif) ligand 14”, “CXCL14 protein” or “CXCL14” refers to a small cytokine belonging to the CXC chemokine family that is also known as BRAK (for breast and kidney-expressed chemokine), which mainly regulates immune cell migration and executes antimicrobial immunity. Yet, the underlying chondrogenic functions and mechanisms are still unknown. In the present invention, one example of CXCL14 used in the present invention isCXCL14. One example of CXCL14 protein (CXCL14-P) is the protein having the amino acid sequence of ITTKSVSRYRGQEH as set forth in SEQ ID NO:1. The CXCL14 proteins also provide the variants thereof, such as CXCL14-PM1 having the amino acid sequence of ITTASVSAYAGQEH as set forth in SEQ ID NO: 4; CXCL14-PM2 having the amino acid sequence of ITTKSVSRYRGQAH as set forth in SEQ ID NO: 5; and CXCL14-PM3 having the amino acid sequence of ITTKSVSRYRGDEH as set forth in SEQ ID NO: 6.

As used herein, the term “CXCL14 gene” or “CXCL14” means the gene coding for the CXCL14 protein, or the CXCL14 mRNA, such as theCXCL14 mRNA referring to NCBI Reference Sequence: NM_004887.5.

As used herein, the term “C—X—C motif chemokine 12”, “CXCL12 protein” or “CXCL12,” as known as stromal cell-derived factor 1 (SDF-1), refers to a chemokine protein that in humans is encoded by the CXCL12 gene on chromosome 10. The CXCL12 proteins belongs to the group of CXC chemokines, whose initial pair of cysteines are separated by one intervening amino acid. One example of CXCL12 protein (CXCL12-P) is the protein having the amino acid sequence of RANVKHLKILN as set forth in SEQ ID NO: 2. The CXCL12 proteins also provide the variants thereof, such as CXCL12-PM1 having the amino acid sequence of AANVAHLAILN as set forth in SEQ ID NO: 7.

As used herein, the term “CXCR4” refers to a protein C—X-C chemokine receptor type 4 (CXCR-4) also known as fusion or CD184 (cluster of differentiation 184), which is a protein in humans encoded by the CXCR4 gene. CXCR4 is present in newly generated neurons during embryogenesis and adult life where it plays a role in neuronal guidance. The levels of the receptor decrease as neurons mature. CXCR4 mutant mice have aberrant neuronal distribution. This has been implicated in disorders such as epilepsy. One example of CXCR4 protein (CXCR4-P) is the protein having the amino acid sequence of MGYGKKLRSMTDKYRL as set forth in SEQ ID NO: 3. The CXCR4 protein also provide the variants thereof, such as CXCR4-PM1 having the amino acid sequence of MGYGAALASMTDAYAL as set forth in SEQ ID NO: 8.

As used herein, the term “CXCR4 agonist” refers to a compound which binds to and activates the CXCR4, including for example, CXCL12 or CXCL14, or a variant, variable domain, functional derivative, or fragment thereof, or a genetically modified recombinant protein or chemically modified protein thereof, which has the same function of CXCL12 or CXCL14.

In one example of the present invention, the CXCR4 agonist is a peptide compound, i.e., a peptide or protein.

As used herein, the term “CXCR4 agonistic activity” refers to the activation of the CXCR4.

In one example of the present invention, the CXCR4 agonist is CXCL14 protein, or a variant, variable domain, functional derivative, or fragment thereof having the same function of CXCL14, a genetically modified recombinant protein or chemically modified protein thereof, or a gene construct for expressions of CXCL14.

In one example of the present invention, the CXCR4 agonist is a CXCL12 protein, or a variant, variable domain, functional derivative, or fragment thereof having the same function of CXCL12, a genetically modified recombinant protein or chemically modified protein thereof, or a gene construct for expressions of CXCL12.

As used herein, the term “extracellular vesicles” or “EVs” refers to lipid bilayer-delimited particles that are naturally released from almost all types of cells but, unlike a cell, cannot replicate. EVs can be divided according to size and synthesis route into exosomes, microvesicles and apoptotic bodies. The composition of EVs varies depending on their parent cells, encompassing proteins (e.g., adhesion molecules, cytoskeletons, cytokines, ribosomal proteins, growth factors, and metabolic enzymes), lipids (including cholesterol, lipid rafts, and ceramides), nucleic acids (such as DNA, mRNA, and miRNA), metabolites, and even organelles. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, cargo, cellular source, and function, leading to a historically heterogenous nomenclature including terms like exosomes and ectosomes.

In the present invention, the chondrogenic differentiation of hBM-MSC was confirmed and the selecting potential genes regulating the induction of chondrogenesis were verified by exploiting microarray gene expressions. Microarray data showed that the (′XCL14 gene, which highly expressed in hBM-MSC during chondrogenesis. CXCL14, that is a novel chemokine, mainly regulates immune cell migration and executes antimicrobial immunity. Yet, the underlying chondrogenic functions and mechanisms are still unknown. It was ascertained in the invention that CXCL14 could enhance chondrogenesis and would correlate with dynamic changes during chondrogenesis, and the mechanisms.

It was demonstrated in the present invention that the human infrapatellar fat-pad MSC (IPFP-MSC) chondrogenesis can be significantly improved by treating the recombinant protein CXCL14 and cartilage regeneration in an ACLT-induced OA mouse model. Furthermore, highly detected chondrogenic genes co-treated with a low dose of TGF-b showed CXCL14 significantly stimulated chondrogenic induction, suggesting that TGF-b downstream signaling might strengthen the CXCL14 in regulating the chondrogenic capacity. CXCR4 serves as a primary receptor for CXCL14 [11], and CXCL12 can also bind to CXCR4 [12], thereby inducing chondrogenesis. In the invention, the functional peptides derived from CXCL14/CXCL12 were synthesized, which reveal a more pronounced potential to augment chondrogenesis compared to the complete sequences of CXCL14/CXCL12.

The present invention initially unveiled the involvement of CXCL14/CXCL12-activated CXCR4 signaling in regulating chondrogenic induction from MSCs. Furthermore, these potential targets demonstrated efficacy in alleviating OA progression induced by ACLT. Thus, our findings suggest that the CXCL14/CXCL12 peptides can influence chondrogenesis induction and may serve as promising candidates for OA therapy. Moreover, we aim to understand the mechanism of CXCR4 signaling activation in regulating chondrogenesis. The results could provide an understanding of the functions and regulation of chondrocyte differentiation by CXCR4 signaling activation and shed light on the identification of new therapeutic targets.

In one particular example of the present invention, the cartilage damage is osteoarthritis (OA).

In the method of the invention, the composition for cartilage regeneration or treatment of cartilage defect disease comprises:

According to the invention, the stem cells can be treated with a CXCR4 agonist to prepare a cell preparation for use in the method for cartilage regeneration. Various types of MSCs can be used, including for example, Wharton's jelly-derived MSCs (WJ-MSCs), infrapatellar fat pad-derived MSCs (IPFP-MSCs), subcutaneous adipose tissue-derived MSCs (SC-MSCs), amniotic fluid-derived MSCs (AF-MSCs), bone marrow-derived MSCs (BM-MSCs), and umbilical cord-derived MSCs (UC-MSCs), which have been investigated for its potential in inducing chondrogenesis. It is ascertained in the present invention that CXCL14, CXCL12, or CXCR4 plays a role in regulating cell differentiation, such as osteoclasts and osteoblasts, was also explored for its potency.

This study was recruited patients aged 50 to 75 undergoing joint replacement surgery, comprising both non-OA and OA individuals (n=6). IPFPs were promptly obtained and processed. All participants were required to sign informed consent forms to utilize their IPFP tissue. Patients with rheumatic diseases treated with immunosuppressive drugs in the past three months were excluded. Approval for the study was granted by the Research Ethics Committee of Far Eastern Memorial Hospital, Taipci (111225-F), Taiwan.

To assess differentiation capacity, single-cell suspensions from cultured IPFP-MSCs and ADSCs were cultivated in chondrogenic medium. Alcian Blue staining was performed on day 7 to evaluate chondrogenic differentiation. The chondrogenic differentiation test was measured gene expressions of ACAN, COL2A1, SOX9, COMP, ITGB1, CXCL14 and SOSTDC1.

Human IPFP-MSCs and ADSCs were expanded in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, cat. no 2145484) with 10% FBS under a 37° C., 5% CO2 atmosphere. MSCs collected and cell extractions were determined gene expression levels of cytokines and chondrogenic markers using a LightCycler® (Roche Applied Science). The fold change in gene expression on the y-axis was calculated based on untreated cell gene expression.

Data were presented as mean±SEM and analyzed using GraphPad Prism software (Version 9.0). One-way ANOVA was calculated P values for multiple comparisons with the indicated post hoc Bonferroni test, utilizing at least three replicates. A non-paired Student's t-test was employed for two-group comparisons, while in vivo data were undergoing analysis using two-way ANOVA.